Bispecific antibody point mutations for enhancing rate of clearance

Abstract

A mutant bispecific antibody that includes (a) a human hinge constant region from IgG having one or more amino acid mutations in the C H 2 domain, (b) two scFvs; and (c) two Fvs has been constructed. This type of antibody displays enhanced clearance, which has been found to be particularly useful in the context of pre-targeting methods.

What is claimed is:  
     
         1 . A mutant bispecific antibody, comprising at least: 
 (a) a human hinge constant region from IgG;    (b) two scFvs; and    (c) two Fvs,    wherein said constant region contains one or more amino acid mutations in the C H 2 domain.    
     
     
         2 . The mutant bispecific antibody of  claim 1 , wherein said scFvs and said Fvs are CDR-grafted murine scFvs and Fvs.  
     
     
         3 . The mutant bispecific antibody of  claim 1 , wherein said scFvs and said Fvs are humanized or human.  
     
     
         4 . The mutant bispecific antibody of  claim 1 , wherein said hinge constant region contains a mutation in which isoleucine at position 253 is replaced with alanine, or amino acids other than leucine, wherein the amino acid replacement enhances blood clearance equal to, or greater than replacement with alanine.  
     
     
         5 . The mutant bispecific antibody of  claim 4 , wherein said Fvs are derived from hMN14-IgG and said scFvs are 734scFv.  
     
     
         6 . The mutant bispecific antibody of  claim 1 , wherein said scFvs bind a monovalent targetable construct.  
     
     
         7 . The mutant bispecific antibody of  claim 1 , wherein said scFvs bind a divalent targetable construct.  
     
     
         8 . The mutant bispecific antibody of  claim 1 , wherein said Fvs bind to an epitope on a target cell.  
     
     
         9 . A method of treating or diagnosing or treating and diagnosing a disease or a condition that may lead to a disease comprising 
 (A) administering to said subject the mutant bispecific antibody of  claim 1 , wherein the Fvs are directed to a marker substance associated with the disease or condition;    (B) optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation; and    (C) administering to said subject a targetable construct comprising a bivalent hapten, wherein both hapten moieties bind to the two scFvs on a single molecule of the mutant bi-specific of  claim 1 , wherein the targetable construct further comprises a diagnostic or therapeutic cation, and/or one or more chelated or chemically bound therapeutic or diagnostic agents.    
     
     
         10 . The method as claimed in  claim 9 , wherein said mutant bispecific antibody is administered before, during or after the administration of at least one therapeutic agent used to treat the disease or condition.  
     
     
         11 . The method as claimed in  claim 9 , wherein said targetable construct comprises an enzyme and said method further comprises administering to said subject 
 a) a prodrug, when said enzyme is capable of converting said prodrug to a drug at the target site; or    b) a drug which is capable of being detoxified in said subject to form an intermediate of lower toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site, or    c) a prodrug which is activated in said subject through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site.    
     
     
         12 . The method of  claim 11 , wherein said prodrug is selected from the group consisting of epirubicin glucuronide, CPT-11, etoposide glucuronide, daunomicin glucuronide and doxorubicin glucuronide  
     
     
         13 . The method of  claim 9 , wherein said diagnostic agent emits 25 to 4,000 keV gamma particles and/or positrons.  
     
     
         14 . The method of  claim 9 , wherein the diagnostic agent is selected from the group consisting of  18 F,  52 Fe,  62 Cu,  64 Cu,  67 Cu,  67 Ga,  68 Ga,  86 Y,  89 Zr  94m Tc,  94 Tc,  99m Tc,  111 In,  123 I,  124 I,  125 I,  131 I,  154-158 Gd,  177 Lu,  32 P, 188Re, and  90 Y or a combination thereof.  
     
     
         15 . The method of  claim 9 , wherein said radioactive isotope is used to perform positron-emission tomography (PET).  
     
     
         16 . The method of  claim 9 , wherein said targetable construct comprises one or more image enhancing agents for use in magnetic resonance imaging (MRI).  
     
     
         17 . The method of  claim 16 , wherein said enhancing agent is selected from the group consisting of Mn, Fe and Gd.  
     
     
         18 . The method of  claim 9 , wherein said targetable construct comprises one or more image enhancing agents for use in ultrasound imaging.  
     
     
         19 . The method of  claim 18 , wherein said targetable construct is a liposome with a bivalent DTPA-peptide covalently attached to the outside surface of the liposome lipid membrane.  
     
     
         20 . The method of  claim 19 , wherein said liposome is gas filled.  
     
     
         21 . The method of  claim 9 , wherein said targetable construct comprises one or more radioactive isotopes useful for killing diseased tissue.  
     
     
         22 . The method of  claim 21 , wherein the energy range of the radioactive isotope is 60 to 700 keV.  
     
     
         23 . The method of  claim 21 , wherein said radioactive isotope is selected from the group consisting of  32 P,  33 P,  47 Sc,  64 Cu,  67 Cu,  67 Ga,  90 Y,  111 Ag,  111 In,  125 I,  131 I,  142 Pr,  153 Sm,  161 Tb,  166 Dy,  166  Ho,  177 Lu,  186 Re,  188 Re,  189 Re,  212 Pb,  212 Bi,  213 Bi,  211 At,  223 Ra and  225 Ac or a combination thereof.  
     
     
         24 . The method of  claim 21 , wherein said targetable construct comprises  10 B atoms, and said method further comprises the step of irradiating said boron atoms localized at said diseased tissue, thereby effecting BNCT of said diseased tissue.  
     
     
         25 . The method of  claim 9 , wherein said therapeutic agent is a drug, toxin, hormone, enzyme, immunomodulator, chelator, boron compound, photoactive agent, dye, or radioisotopes.  
     
     
         26 . The method of  claim 21 , wherein said targetable construct comprises one or more toxins.  
     
     
         27 . The method of  claim 26 , wherein said toxin is selected from the group consisting of ricin, abrin, ribonuclease, DNase 1, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin or a combination thereof.  
     
     
         28 . The method of  claim 9 , wherein said targetable construct comprises one or more drugs.  
     
     
         29 . The method of  claim 28 , wherein said drug is selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, antagonists, endostatin, taxols, camptothecins, doxorubicins and their analogs, and a combination thereof.  
     
     
         30 . The method of  claim 9 , wherein the targetable construct comprises one or more agents for photodynamic therapy.  
     
     
         31 . The method of  claim 30 , wherein said agent for photodynamic therapy is a photosensitizer.  
     
     
         32 . The method of  claim 31 , wherein said photosensitizer is selected from the group consisting of benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex).  
     
     
         33 . The method of  claim 9 , wherein said targeted tissue is a tumor.  
     
     
         34 . The method of  claim 11 , wherein said targeted tissue is a tumor.  
     
     
         35 . The method of claims  33  or  34 , wherein said tumor produces or is associated with antigens selected from the group consisting of colon-specific antigen-p (CSAp), carcinoembryonic antigen (CEA), CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66a-e, CD74, CD75, CD80, CD126, B7, HLA-DR, Ia, Ii, HM1.24, MUC 1, MUC2, MUC3, MUC4,Tag-72, PSMA, EGP-1, EGP-2, PSA, AFP, HCG, HCG-beta, PLAP, PAP, histone, tenascin, VEGF, P1GF, S10O, EGFR, insulin-like growth factor, HER2/neu, organotropic hormones, oncogene products, and cytokeratin.  
     
     
         36 . The method of claims  9  or  11 , wherein the mutant bispecific antibody incorporates the Fv of a Class III anti-CEA antibody.  
     
     
         37 . The method of claims  9  or  11 , wherein the mutant bispecific antibody incorporates the scFv of Mab 679.  
     
     
         38 . The method of claims  9  or  11 , wherein said disease is an immune dysregulation disease, an autoimmune disease, organ graft rejection, cardiovascular disease, neurological disease or graft vs. host disease.  
     
     
         39 . The method of  claim 38  wherein said autoimmune disease is selected from the group consisting of acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, parnphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.  
     
     
         40 . The method of claims  9  or  11 , wherein said disease caused by a fungus, virus, parasite or bacterium, and the Fv of the mutant bispecific targets the fungus, virus, parasite, or bacterium.  
     
     
         41 . The method of  claim 40 , wherein said virus is selected from the group consisting of human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, polio virus, human serum parvo-like virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus and blue tongue virus.  
     
     
         42 . The method of  claim 40 , wherein said bacterium is selected from the group consisting of  Anthrax bacillus, Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis , Pneumococcus, Hemophilis influenzae B,  Treponema pallidum , Lyme disease spirochetes,  Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis  and Tetanus toxin.  
     
     
         43 . The method of  claim 40 , wherein said pathogen is a protozoan.  
     
     
         44 . The method of  claim 43 , wherein said protozoan is selected from the group consisting of  Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Onchocerca volvulus, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus  and  Mesocestoides corti.    
     
     
         45 . The method of  claim 40 , wherein said parasite is a helminth or a malarial parasite.  
     
     
         46 . The method of  claim 40 , wherein said bacterium is mycoplasma.  
     
     
         47 . The method of  claim 46 , wherein said mycoplasma is selected from the group consisting of  Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarum , and  M. pneumoniae.    
     
     
         48 . The method of  claim 40 , wherein the fungus is selected from the group consisting of  Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis , and species of Candida.  
     
     
         49 . The method of claims  9  or  11 , wherein the tissue is normal ectopic tissue.  
     
     
         50 . The method of  claim 49 , wherein said normal tissue is tissue from the ovary, thymus, parathyroid, bone marrow, or spleen.  
     
     
         51 . The method of claims  9  or  11 , wherein said subject is mammalian.  
     
     
         52 . The method of claims  9  or  11 , wherein said mammalian subject is a human or primate.  
     
     
         53 . The method of  claim 51 , wherein said mammalian subject is selected from the group consisting of rodents, lagamorphs, bovines, ovines, caprines, porcines, equines, canines, felines, domestic fowl, ungulates, and bear.  
     
     
         54 . The method of  claim 9 , wherein the application is for intraoperative diagnosis to identify diseased tissues.  
     
     
         55 . The method of  claim 9 , wherein the application is for endoscopic diagnosis to identify diseased tissues.  
     
     
         56 . The method of any one of claims  9 - 55  wherein a second therapeutic agent is administered before, concurrently, or after the prescribed diagnosis or treatment.  
     
     
         57 . The method of  claim 56 , wherein the second therapeutic agent is a drug, naked antibody, immunomodulator, or antibody conjugate bearing a drug, radioisotope, immunomodulator or toxin.  
     
     
         58 . A kit useful for treating or identifying diseased tissues in a subject comprising: 
 (A) the mutant bispecific antibody of  claim 9;     (B) optionally, the clearing agent of  claim 9;  and    (C) the targetable construct of  claim 11 .    
     
     
         59 . A kit useful for treating or identifying diseased tissues in a subject comprising: 
 (A) the mutant bispecific antibody of  claim 11;     (B) optionally, the clearing agent of  claim 11;     (C) the targetable construct of  claim 11;  and    (D) the prodrug of  claim 11 .    
     
     
         60 . The method of  claim 25 , wherein said immunomodulator is selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof.  
     
     
         61 . The method of  claim 60 , wherein said lymphotoxin is tumor necrosis factor (TNF).  
     
     
         62 . The method of  claim 60 , wherein hematopoietic factor is interleukin (IL).  
     
     
         63 . The method of  claim 60 , wherein said colony stimulating factor is granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF).  
     
     
         64 . The method of  claim 60 , wherein said interferon is interferon-α, -β or -γ.  
     
     
         65 . The method of  claim 60 , wherein said stem cell growth factor is S1 factor.  
     
     
         66 . The method of  claim 25 , wherein said immunomodulator is IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, interferon-γ, TNF-α or a combination thereof.  
     
     
         67 . The method of  claim 38 , wherein said neurological disease is Alzheimer's Disease.